DNA gyrase introduces negative supercoils into DNA to maintain topological homeostasis. The genes encoding gyrase, gyrB and gyrA, form a dicistronic operon in Mycobacterium tuberculosis (Mtb) and other actinobacteria. Earlier work indicated that DNA relaxation stimulates the expression of the gyr genes, a phenomenon termed relaxation-stimulated transcription (RST). The present study addresses the underlying mechanism of gyr operon regulation. The operon is regulated by overlapping and divergently oriented promoters located upstream of gyrB. The principal promoter, P-gyrB1, drives transcription of the operon, while a weak "reverse" promoter, P-gyrR, transcribes in opposite direction. We demonstrate that P-gyrR plays a role in fine tuning gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not found in Mtb. In vitro transcription assays showed that RT at P-gyrR depended on the negatively supercoiled state of the DNA template. The principal promoter, P-gyrB1, was also sensitive to DNA supercoiling, but it was stimulated by DNA relaxation. Moreover, RNA polymerase binding to the promoter was efficient at P-gyrB1 when template DNA was relaxed, whereas binding to P-gyrR was preferred when DNA was supercoiled. Thus, a collaboration between RST and RT governs the regulation of the gyr operon; the differing sensitivity of the two overlapping promoters to superhelix density explains how gyrase expression responds to changes in supercoiling to determine the efficiency of transcription initiation. (C) 2018 Elsevier Inc. All rights reserved.